Methods and compositions for the introduction of molecules into cells

ABSTRACT

The present invention is directed to the introduction of molecules, including nucleic acids, carbohydrates, plant growth regulators and peptides into cells and tissues. The present invention is also directed to media and methods for enhancing embryogenic callus production of elite lines of soybean.

CROSS REFERENCE

This application is a divisional of U.S. patent application Ser. No.09/724,265 filed Nov. 28, 2000, now U.S. Pat. No. 6,809,232, which is acontinuation-in-part of U.S. patent application Ser. No. 09/450,226,filed Nov. 29, 1999, now abandoned.

BACKGROUND OF THE INVENTION

The present invention is directed to a method for the introduction ofmolecules into cells, including but not limited to bacterial and plantcells. The molecules which are introduced by the method of the inventioninclude, without limitation, nucleic acids, carbohydrates, plant growthregulators and peptides. The method of the invention is further directedto the transformation of bacteria and plant cells and tissues and to theresulting transformed cells and tissues. The present invention is alsodirected to a method and medium for initiating more rapid and uniformgrowth of embryogenic callus, specifically the growth of soybeanembryogenic callus.

The publications, patents and other materials used herein to illuminatethe background of the invention, and in particular cases, to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

Delivery of Molecules to Cells

Small and large molecules can be efficiently delivered to cells withoutcell walls by electric pulsing (Dagher et al., 1991), electroporation(Fromm et al., 1986) or through mediation by polyethylene glycol (Klebe,R. J., et al., 1983). These technologies, however, are of limited usewith plants due to the presence of the plant cell wall. Other methodshave been developed specifically for DNA delivery to plant cells, suchas particle bombardment (Sanford et al., 1987), silicon carbide whiskertechnology (Kaeppler et al., 1990), and electroporation (D'Halluin etal., 1992). However, each of these delivery methods has significantlimitations. For example, particle bombardment, while reported effectivefor transformation of some plant cells, typically relies onprecipitation of DNA molecules onto the surface of inert carrierparticles prior to delivery. As a result, this requirement limits theusefulness of the technology for delivery of molecules such as proteins.In fact, there are no reports of effective delivery of proteins to plantcells using particle bombardment.

Silicon carbide whisker technology is reported to be much less efficientthan particle bombardment for DNA delivery to plant cells and has beenshown to be effective only in one cell type and single genotype of corn(Frame et al., 1994). Delivery of DNA to cells via electroporation hasbeen described (D'Halluin et al., 1992; Laursen et al., 1994), however,this technology is ineffective for most cell types and there are veryfew reports of its successful use in plant transformation research.Furthermore, there are no known reports of its use to deliver proteinsand other large molecules to the cells of higher plants.

Microinjection has been used to introduce proteins (Neuhaus et al.,1987) and DNA (Neuhaus, et al., 1987; U.S. Pat. No. 4,743,548) intoplant cells. The principal limitations of microinjection are that it isextremely time-consuming and possible only with cells that can beisolated and handled as single entities. For these reasonsmicroinjection has not been the method of choice for the transformationof any plant species where the goal is to produce genetically modifiedgermplasm.

Current aerosol beam technology has been reported to be capable oftransforming the chloroplast genome of Chlamydomonas, a unicellular,green alga (Mets, U.S. Pat. No. 5,240,842). Chlamydomonas chloroplasttransformation can be considered a special situation since thechloroplast of Chlamydomonas is large, filling the entire cell of thetypically 10 micron size organism. However, nuclear transformation wasnot reported by Mets and the only organism reported transformed wasChlamydomonas. Furthermore, in the eight years since the technology wasfirst published, aerosol beam technology has not been reported to effectnuclear transformation of any species. Sautter et al. (1991) and U.S.Pat. No. 5,877,023, describe a technology which combines aspects of theaerosol beam and particle bombardment. Transformation with thetechnology reported by Sautter, et al., depends upon the inclusion ofgold carrier particles of 1 micron diameter. There have been no otherreports of the successful use of this technology.

As those of ordinary skill in the art recognize, it would be desirableto introduce a range of molecules including proteins and othermacromolecules into plant and bacterial cells. This would allow, amongother possibilities, the pursuit of pioneering studies in functionalgenomics. It is clear therefore that there is a need to improve aerosolbeam technology to the point where it can be used routinely to effectnuclear transformation of important crop species such as corn andsoybean and also to introduce other large macromolecules into cells. Themethod of the present invention solves this need.

Methods of Tissue Culturing

Cells which undergo rapid division and are totipotent are generallyregarded as highly suitable targets for introduction of DNA as a firststep in the generation of transgenic plants. Undifferentiated cells intissues, such as meristematic tissues and embryogenic tissues areespecially suitable. In general, cells of elite lines of crop plants aredifficult to grow in culture. Specifically, cell division afterintroduction of nucleic acid is difficult to sustain and thereforeselection of transformed cells often proves impossible.

Typically, embryogenic callus of soybean is cultured on highconcentrations of 2,4-D (Ranch et al., 1985). However, even with highconcentrations of 2,4-D in the culture medium, many cultivars do notproduce sufficient embryogenic callus for transformation experiments.Specifically, there are no reports of high frequency initiation ofcallus from immature embryos or other tissue of elite soybean lines.

The useful lifetime of a soybean variety in the marketplace is usuallyaround three years. This does not allow time for the backcrossing oftransgenes into new and elite varieties from lines that are not elite,since by the time this could be accomplished, new varieties would havereplaced those chosen as the recurrent parents in the backcrossingprogram. Furthermore, problems with loss of yield are commonlyencountered when transgenes are introduced into elite material fromnon-elite transformants (Minor, 1998; Oplinger, 1998). Therefore,improved culture media which are capable of supporting rapid and uniformgrowth of a range of soybean germplasm would represent a significantadvance in the art. Such an improved media are described herein.

SUMMARY OF THE INVENTION

The present invention is directed to a method for the introduction ofmolecules into cells, including but not limited to bacterial and plantcells. The molecules which are introduced by the method of the inventioninclude, without limitation, nucleic acids, carbohydrates, plant growthregulators and peptides. The method of the invention is further directedto the transformation of bacteria and plant cells and tissues and to theresulting transformed cells and tissues. The present invention is alsodirected to a method and medium for initiating more rapid and uniformgrowth of embryogenic soybean callus, specifically the growth of elitelines of soybean.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a schematic illustration of the aerosol beam apparatus of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

Using the method of the present invention it is possible to introducemolecules, including macro molecules, into cells without the need forprecipitation of the molecules onto carrier particles, and therefore, itis not necessary to include protocols, such as precipitation, in orderto associate the molecules with carrier particles. The present inventionis especially useful for introducing peptides into large numbers ofcells allowing for studies in such areas as functional genomics. Thepresent invention can also be used to accomplish nuclear transformationof cells including but not limited to bacteria, and mono- anddicotyledonous plants. More particularly, elite germplasm of crop plantspecies can be transformed using the present invention. The presentinvention is further directed to media and methods for initiating rapidand uniform growth of elite lines of soybean, for example, transformedelite germplasm.

DEFINITIONS

The present invention employs the following definitions:

“Aerosol droplets” refer to droplets or particles, wet or dry, dispersedin a gas.

“Callus” refers to an undifferentiated mass of cells or tissue in vitro.

“Carrier particles” refer to gold or tungsten or other heavy metalparticles ranging in size from 0.1 micron to 4 microns which are used tointroduce molecules into cells.

“Continuous Targeting” refers to the delivery of aerosol droplets in acontinuous stream toward a target.

“Elite Line” refers to a genetic line used in a product, or in theimmediate (within three years) development of a product.

“Embryogenic Callus” refers to tissue composed of large numbers ofsomatic embryos or embryo-like structures.

“Exogenous Gene”, “Exogenous DNA” and “Exogenous Nucleotide Sequence”refer to any gene, DNA or nucleic acid segment that is introduced into arecipient cell, regardless of whether a similar gene may already bepresent in such a cell.

“Germplasm” refers to varieties of genetic types within a species.

“Genotype” refers to the genetic identity of an organism.

“Microflow Nebulizer” refers to any device that creates an aerosol whenprovided with a sample flow rate, with or without use of a syringe pump,of between about 1 μl/minute and about 500 μl/minute.

“Nucleotide sequence” refers to a naturally occurring or non-naturallyoccurring nucleic acid, either isolated, synthesized or the result ofgenetic engineering techniques.

“Phytic Acid” refers to inositolhexaphosphoric acid.

“Stage” refers to the platform on which target cells may be placed inthe method of the invention.

“Target Surface” refers to the cells comprising the uppermost layer ofcells or tissue that is first encountered by the stream of aerosoldroplets produced by the aerosol beam apparatus.

“Transformation” refers to the acquisition of new genetic codingsequences by the incorporation of an exogenous nucleotide sequence.

“Transgenic” and “Transformed” refers to organisms into which exogenousnucleotide sequences are integrated.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, and immunology. See, e.g.,Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992;Glover, 1985; Anand, 1992; Guthrie and Fink, 1991; Gelvin et al., 1990;Grierson et al., 1984.

Method of Delivering Molecules to Cells

The method of the present invention offers a number of advantages overcurrent methods of DNA delivery. Aerosol beam technology of theinvention employs the jet expansion of an inert gas as it passes from aregion of higher gas pressure to a region of lower gas pressure througha small orifice. The expanding gas accelerates aerosol dropletscontaining the molecules to be introduced into a cell or tissue. Thesize of the droplet is of particular importance when molecules are to beintroduced into small cells or cellular organelles, for example, cellsless than about 20 microns. The importance of droplet size has beendemonstrated with particle bombardment technology where particles ofgreater than 1 micron in diameter were shown to be unsuitable forintroducing DNA into cells of 10 to 20 microns in diameter (Klein, etal., 1988(a); Klein, et al., 1988(b) due to the damage produced byparticles of such size. On the other hand, large particles have beenreported to be more likely to penetrate the cells (U.S. Pat. No.5,877,023).

Acceleration of droplets of a DNA containing solution by jet expansionis the subject of U.S. Pat. No. 5,240,842 to Mets which is hereinincorporated by reference. The only successful transformation reportedby Mets is with droplets of 2 micron diameter. Droplets of this sizecould be expected to destroy cells such as bacteria which are typicallyno larger than 2 microns. There are no examples of successful use ofdroplets smaller than 2 microns in diameter described in the patent toMets. The diameter of a substantial portion of the aerosol dropletsgenerated by the method of the invention are believed to be less than0.1 microns at the point of impact with the target cells. This issupported by the ability to routinely and efficiently transformbacterial cells of approximately 1 to 2 microns in length using themethod of the invention. Further support is found in the expectationthat droplets larger than 0.1 micron in diameter are unlikely to be ableto enter a rod-shaped or rectangular cell of 1 to 2 microns in lengthwithout destroying the cell.

Particle bombardment, as practiced using the Dupont PDS-1000, differsfrom aerosol beam technology in part because it relies on accelerationprovided by a single burst of helium gas pressure. Viewed under ascanning electron microscope, the particles from the Dupont gun range insize from 0.1 microns up to 4 microns in diameter (using M5 tungstenparticles with an average diameter of 0.4 microns).

DNA has been introduced into bacterial cells using polyethylene glycol(Klebe et al., 1983), electroporation (Bonmassie et al., 1990),treatment with divalent cations (Hanahan, 1983), and particlebombardment (Smith et al., 1992). Smith et al. (1992), considerconvenience to be one of the main advantages of biolistic transformationover electroporation. Nevertheless, to achieve efficient transformationusing the biolistic process, treatment of bacterial cells with anosmoticum is required as is careful control of the relative humidity atwhich the particles were stored prior to bombardment. Bacterialtransformation using the method of the present invention is much moreefficient than biolistic-mediated transformation and requires no specialpretreatments.

Based on successful and efficient transformation of bacteria using themethod of the present invention, it is believed that a substantialnumber of the aerosol droplets produced are less than 0.1 micron indiameter at the point of impact with the target cells. DNA carried inaerosol droplets of this small size penetrates cells only because of thespeeds attained by the aerosol droplets. Speeds achieved by the aerosolbeam method of the invention are supersonic and can reach 2000meters/second. By contrast, top speed achieved by the particle gun is200 meters/second.

In part, because aerosol droplets generated by the present invention areso much smaller than the particles produced by the prior art, thepresent invention is superior in delivering molecules to small cells,for example cells less than 20 microns in diameter. Many animal, plantand microbial cells are in this size range. Entry of micron-sizeparticles into cells of this size can be expected to cause significantdamage. The very small aerosol droplets produced by the method of thepresent invention are also advantageous for chloroplast transformation,particularly in monocots and graminaceous embryogenic cell culturescontain proplastids (the target for chloroplast transformation) that aretypically less than 1 micron in diameter (Bilang and Potrykus, 1998).

Another advantage of the present invention is that it permits betterquantification of DNA delivery to cells than is possible with particlegun technology. This is because no precipitation or coating of DNA ontocarrier particles is required with the method of the invention,procedures which introduce variability into the DNA delivery process.

As a further embodiment of the present invention, molecules other thanDNA can be readily introduced into cells, either alone or in combinationwith DNA. Inclusion of molecules other than DNA introduced at the sametime as DNA could improve integration and increase the number ofselectable transformants. By contrast, effective delivery of moleculescan be achieved with particle bombardment only after first coating theparticles with the substance to be delivered. In those cases where it isnot possible or desirable to use coated particles, the aerosol beam maybe the most effective way to deliver chemicals directly to the interiorof cells on a large scale.

An additional advantage of the present invention is that DNA isdelivered as a stream of aerosol droplets emerging through a smallorifice (continuous targeting). This targeting can continue for as longas the target tissue can survive being held in a vacuum. In the courseof delivery the droplet stream can then be precisely targeted. Thisfurther distinguishes aerosol beam technology from particle bombardmentas currently practiced where all DNA-coated particles are delivered tocells in a single blast resulting in a shot pattern covering an area ofseveral centimeters in diameter. A hybrid of the aerosol beam andparticle bombardment methods (Sautter, et al.; U.S. Pat. No. 5,877,023)is reported to deliver a solution of DNA and 1 micron diameter goldcarrier particles in interrupted blasts, thus continuous targeting oftissue is not possible with this method. Continuous targeting alsoallows for the identification of an area of tissue (such as the apex ofa shoot meristem) and its positioning under the beam to ensure focuseddelivery of aerosol droplets to the tissue. Furthermore, repeated blastswhich are delivered with particle bombardment and the method of Sautter,et al., can be expected to result in severe and extensive tissuedestruction. The ability to continuously target cells or tissues makesthe aerosol beam clearly superior to other direct DNA delivery methodsincluding particle bombardment.

The aerosol beam of the present invention differs from U.S. Pat. No.5,240,842 in numerous respects. First, Mets does not include anypositive pressure entrainment airflow to guide or focus the aerosolbeam. Second, Mets includes a vent in the compressed gas path to allowrelease of excess aerosol. This results in wasted compressed gas andaerosol material. The vent is required in Mets because of the high flowrates used. Third, the nebulizer used by Mets is a type used ininhalation therapy and is described as of the Lovelace design. Thisnebulizer is a single use disposable unit that generates aerosoldroplets with median mass diameters in the range of 2 microns.Commercially available nebulizers such as HEN or MCN100 were used inexample. However, any microflow nebulizer, as defined herein, can beused in the practice of the present invention. Unexpectedly, use ofmicroflow nebulizers cacilitated insertion of molecules into cells.Fourth, the preferred embodiment of the present invention includes asyringe pump which regulates the flow rate of the sample to thenebulizer. Fifth, droplets of the size described by Mets would be toolarge to deliver DNA or any other molecule to most bacteria or plantcells and yet allow survival of these cells.

Transformation of Plant Cells and Tissues

Introduction of DNA and other molecules such as proteins into plantcells by the method of the present invention is exemplified bytransformation of corn (monocotyledonous plants) and soybean(dicotyledonous plants). Briefly, the transgenic plants of thisinvention may be produced by (I) culturing a source of cells, (II)optionally, pretreating cells to yield tissue with increased capacityfor uptake and integration by aerosol beam technology, (III)transforming said tissue with an exogenous nucleotide sequence by theaerosol beam method of the invention, (IV) optionally, identifying orselecting for transformed tissue, (V) regenerating transgenic plantsfrom the transformed cells or tissue, and (VI) optionally, producingprogeny of said transgenic plants.

Method of Culturing Cells

Corn Cell Cultures.

The corn cells which may be used as starting materials in the presenttransformation process include elite inbred lines of corn. For example,embryogenic callus and immature embryos of Stine Inbred 963 were usedfor both transient expression experiments and the production of stablytransformed callus, embryos and plants. Other cells may be used,including those derived from meristems. These meristems are found, forexample, in juvenile leaves, immature tassels, immature and matureembryos and coleoptilar nodes. While the method of the present inventioncan be applied to any corn cells from which fertile plants can berecovered, cell cultures derived from immature embryos or embryogeniccallus have been utilized herein for purposes of example.

Methods of preparing and maintaining corn cells are well known in theart (Duncan et al., 1985). Typically, cultures are prepared fromimmature maize embryos which have been removed from the kernels of anear when the embryos are about 1-2 mm. in length. The scutellum ofimmature embryos can be stimulated to give rise to embryogenic callusfrom which plants can be regenerated. Embryogenic callus can also beobtained from the developing reproductive organs of a corn plant.Exemplary methods for isolating immature embryos from corn are describedby Green and Phillips (1976).

In a preferred embodiment of the invention, the embryos were placed onculture medium, for example, DN62AG (Table 1), under aseptic conditions.This culture medium, DN62AG, has been described in U.S. Ser. No.09/203,679, filed December, 1998, incorporated herein by reference. Ithas been discovered that immature embryos incubated for approximately 2to 3 days on DN62AG medium, after dissection and prior to beaming, showimproved survival. This, in turn, improves the efficiency with whichtransformants can be recovered. The embryogenic callus cultures areroutinely maintained on stock culture medium, preferably on DN62(Table 1) for ten-day periods between transfers.

Soybean Cell Cultures.

Soybean cells which can be used as starting materials in the method ofthe invention include cell cultures and explants containing meristematictissue from which plants can be regenerated. Exemplary of cells whichare appropriate are embryogenic callus of Stine 13404-TT (Examples 10).

Conventional methods of preparing and maintaining embryogenic soybeancultures are described by Ranch et al. (1985). In one embodiment of theinvention, the medium of Ranch et al (1985), can be modified by theinclusion of one or more of four constituents (Example 8). Immaturecotyledons about 1 mm in length were used as the initial explants andwere placed on culture media under sterile conditions.

In another embodiment of the present invention, a novel culture mediamay be used to stimulate high frequency production of embryogenicsoybean callus. Improvement varied with the genotype being cultured. Thelength of time required for a culture passage was unexpectedly reducedto two weeks with the use of this novel medium as compared to four weekstypical with other media. The inclusion of one or more of four mediaconstituents, coconut water, myoinositol, phytic acid and inorganicphosphate concentration, enhanced embryogenic callus production andallowed significant improvements to be made to transgenic cloneproduction in terms of number of clones recovered, embryo morphology,and reduction in the time needed to identify the clones and regenerateplants from them. The medium of Ranch et al., 1985 (referred to hereinas B1-30) was used as the basal medium. An example of the medium of thepresent invention is B1-30 3Co5My0.25PA0.5K (footnote, Table 5).Although this medium is a preferred embodiment of growth medium, otherconventional media may be utilized in the practice of the invention.

Pretreatment of Cells

In another embodiment of the present invention, pretreatment of thecells may be carried out in order to increase nucleic acid deliveryusing the method of the invention. For example, corn cells can beosmotically stressed to improve DNA delivery while preserving cellviability. Possible methods of osmotic stress include those conventionalmethods known in the art, for example, Russell et al., 1992. As apreferred embodiment of the invention, a novel medium designed toprovide osmotic stress referred to as DN62OSM was used (Example 2, Table1). The duration of exposure to osmotic stress may range from about 45minutes up to about 24 hours on this medium, with a preferred durationof about 45 minutes to about one hour. In the case of soybean cells anosmotic pretreatment was not employed.

Introduction of Nucleic Acid into Cells

One embodiment of the present invention is directed to the introductionof a nucleotide sequence into plant cells via aerosol beam technology.Once a desired nucleotide sequence has been synthesized or cloned, andengineered, it is necessary to integrate it into the genome of a plantof interest so that it is stably inherited by progeny of the transformedplant. Following introduction of foreign sequence into target cells andsubsequent cell division, selection is applied to identify those cellsin which integration and expression of the sequence is occurring. Stabletransformation involves the integration of functional genetic sequencesinto the genome so that the integrated sequences are passed on to andare present in the transformed plants. Any procedure which could enablethe stable integration of nucleic acids would greatly improvetransformation protocols. In contrast, transient transformation resultsin eventual loss of the sequence and, therefore, transient methods areof little use in generating transgenic plants, although they may be ofuse in the optimization of conditions for stable transformation and inevaluation of gene expression.

An embodiment of the aerosol beam apparatus of the present invention isshown in FIG. 1. The apparatus includes a pressurized gas supply 10, avacuum chamber 13, and an entrainment housing 11.

The pressurized gas supply 10 may contain a pressurized propellant gassuch as, for example, helium. The pressurized gas supply 10 is connectedto a microflow nebulizer conduit 7 and is also connected to anentrainment tube 5.

The entrainment housing 11 has an interior, and located within theinterior is all or a portion of a microflow nebulizer 8. The entrainmenthousing 11 interior communicates with the pressurized gas supply 10 viathe entrainment tube 5, which may include a pressure regulator 20. Themicroflow nebulizer 8 communicates with the pressurized gas supply 10via the microflow nebulizer conduit 7, which may include a filter 16 anda pressure regulator 19. A single regulator may be employed if theentrainment gas pressure can be the same as the nebulizer gas pressure.The entrainment housing 11 may also include a temperature controller 17which controls the temperature in the entrainment housing 11 to a rangeof about 32 degrees to about 80 degrees Centigrade. A pressure gauge 6is connected to the entrainment housing 11 and may be used to controlthe pressure regulator 20 to set the entrainment air pressure and flow.The entrainment housing 11 may include a nucleospot 9. The nucleospot 9may be used to reduce electrostatic charges created by the moving gas.

The microflow nebulizer 8 is an aerosol nebulizer that is fed both asample material and a pressurized gas. The sample material may be fed tothe microflow nebulizer 8 by a pump or other suitable means. In themicroflow nebulizer 8, the pressurized gas forces the sample materialthrough a small orifice in the nebulizer, where the pressurized gasconverts the sample material into aerosol droplets. The aerosol dropletsare then carried by the resulting beam of gas to the nozzle, throughwhich the aerosol droplets greatly accelerated.

A sample material supply conduit 4 is connected to the microflownebulizer 8. The sample material supply conduit 4 may be furtherconnected to a pump 2 and filter 3. In a preferred embodiment, the pump2 is a syringe pump and includes a plastic syringe 1 holding a quantityof an sample material.

In addition to the aerosol beam, pressurized helium flows into theentrainment housing 11 through the entrainment tube 5. This entrainmentgas flow also has a velocity and moves substantially parallel to theaerosol spray, and serves to entrain the aerosol spray and focus it onthe way to the target.

The vacuum chamber 13 includes a nozzle 12, a vacuum pump 15, and a door22. The vacuum chamber 13 may additionally include a stage 14, a vacuumgauge 21, and a stage switch 18. The nozzle 12 further guides theaerosol beam as it approaches the stage 14 (on which the target to beinjected rests). The nozzle 12 includes an orifice, with the orificesize ranging from about 200 microns to about 500 microns. The orificesize is preferably 300-330 microns. The combination of the pressurizedgas in the entrainment housing 11 and the partial vacuum in the vacuumchamber 13 accelerates the aerosol droplets that impact the targetcells.

The stage 14 may be a movable stage wherein the target may be movedrelative to the incoming aerosol spray so that a controlled area may beimpacted with the sample material. The stage switch 18 may be used tocontrol movement of the stage 14. The vacuum gauge 21 may be used tomonitor and control the vacuum level in the vacuum chamber 13.

The aerosol can be produced by various microflow nebulizers known in theart, such as the HEN from J. E. Meinhard Associates, Inc., or the MCN100style M-4 nebulizer from Cetac Technologies, Inc., although othermicroflow nebulizers may also be used. The preferred nebulizer describedin U.S. Pat. No. 5,240,842 was one typically used in inhalation therapyand was described as being of the Lovelace design which is a single-usedisposable unit that generates aerosol droplets with median massdiameters in the range of 2 microns.

The nebulizing gas can be selected from those inert gases known in theart, preferably high purity compressed helium. The gas is regulated andfiltered. The entrainment gas can be high purity compressed helium,filtered or not. The entrainment tube or housing may contain anucleospot to reduce electrostatic charges and can be maintained at atemperature of between about 32 and 80° C. by temperature controllers.The sample flow rate may be set at from about 1 to about 1200 μl/min.

The method of the present invention differs from the several embodimentsdescribed in Mets (U.S. Pat. No. 5,240,842) wherein a common feature wasthe presence of a vent to allow release of excess aerosol which was theresult of the high flow rates that were used. It has been unexpectedlydiscovered that high efficiency microflow nebulizers, such as the HENand MCN 100, provide adequate aerosol droplet production when using verylow (1 to 350 μl/min) flow rates. Venting of excess aerosol is thereforenot necessary with the method of the invention. Furthermore, the aerosoldroplets produced by the microflow nebulizers in the method of theinvention are much smaller at the point of impact with the target thanthe 2 micron diameters preferred by Mets. Small droplet size can bemaintained in the method of the invention by the use of helium as thenebulizing gas although other means known in the art may be used.

Improved efficiency of transformation is possible using the method ofthe invention which employs the routine production of small aerosoldroplets traveling at supersonic speed. The correlation between thereduced droplet size and improved efficiency was unexpected. It waspreviously reported in U.S. Pat. No. 5,877,023, in regard to particlesize, that large particles are more able to penetrate the cells thoughuse of small particles is more favorable for the survival of cells (Col.7, L. 14-18). The method of the invention also improves upon existingtechnology by enabling the routine transformation of bacteria.

The chamber vacuum can be maintained at from about 26 to about 30 in. Hgthroughout a given run with use of a vacuum pump. Water may be placed inthe vacuum chamber to prevent loss of moisture from the target tissue. Asyringe needle cut off just proximal to the plastic holder can be usedfor the nozzle. Nozzle diameters of from about 200 to about 500 micronscan be used. The target tissue can be placed in the center of an agarplate below the nozzle tip. The stage movement can be adjusted toachieve the desired result.

Briefly, treatment of target tissue with the aerosol beam apparatus maybe performed as follows: (1) place tissue on target surface, on thestage; (2) start the vacuum pump; (3) set the nebulizing gas pressure;(4) set the entrainment gas pressure; (5) start the syringe pump; (6)start the movement of the stage and let it run while the aerosolparticles suspended in the inert gas impact the target tissue. Deliverycan continue for as long as the target tissue can survive being held ina vacuum.

In order to successfully produce stably transformed plants by aerosolbeam technology, four requirements must be met: (1) the target cellsmust remain viable; (2) the target cells must be able to take up therecombinant nucleic acid at high enough frequencies to insure the stabletransformation of a useful number of cells; (3) once transformed, therecipient cells must be able to maintain cell division and regenerativecapacity throughout the selection process in order to confirm andidentify stably transformed cells; and (4) the transformed regeneratedplants must be able to express the recombinant nucleic acid.

Utilizing the method of the present invention, accurate and extensivetissue targeting can be achieved with any explant, including immatureembryo, immature tassel, section of leaf or root, anther, pollen andmeristem cells of corn, and meristem and somatic embryo cells ofsoybean. For example, in the case of corn, pollen, as well as itsprecursor cells, microspores, may be capable of functioning as recipientcells for nucleic acid delivery, or as vectors to carry foreign nucleicacid for incorporation during fertilization. The continuous targetingwhich is possible with the method of the invention enables flexibilityand accuracy in delivering nucleic acid to target cells. Individualimmature corn embryos can be targeted so that only particular regions ofthe scutellum are subjected to the aerosol beam or, alternatively, it ispossible to deliver nucleic acid to the entire surface area of thescutellum with the aerosol beam.

Examples of genes useful for expression in transformed plant cells areknown in the art. More particularly, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Suchgenes include, but are not limited to, those described herein.

Genes That Confer Resistance or Tolerance to Pests or Disease

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance (R)gene in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. Examples of such genes include, the tomato Cf-9 genefor resistance to Cladosporium fulvum (Jones et al. (1994)), the tomatoPto gene, which encodes a protein kinase, for resistance to Pseudomonassyringae pv. tomato (Martin et al. (1993)), and the Arabidopsis RSSP2gene for resistance to Pseudomonas syringae (Mindrinos et al. (1994)).

(B). A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon, such as, a nucleotide sequence ofa Bt δ-endotoxin gene (Geiser et al. (1986)). Moreover, DNA moleculesencoding δ-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), under ATCC accession numbers. 40098, 67136,31995 and 31998.

(C) A lectin, such as nucleotide sequences of several Clivia miniatamannose-binding lectin genes (Van Damme et al. (1994)).

(D) A vitamin binding protein, such as avidin and avidin homologs whichare useful as larvicides against insect pests. See U.S. Pat. No.5,659,026.

(E) An enzyme inhibitor, e.g., a protease inhibitor or an amylaseinhibitor. Examples of such genes include a rice cysteine proteinaseinhibitor (Abe et al. (1987)), a tobacco proteinase inhibitor I (Huub etal. (1993)), and an α-amylase inhibitor (Sumitani et al. (1993)).

(F) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. Examples of such genesinclude, an insect diuretic hormone receptor (Regan (1994), anallostatin identified in Diploptera puntata (Pratt (1989)),insect-specific, paralytic neurotoxins (U.S. Pat. No. 5,266,361).

(G) An insect-specific venom produced in nature by a snake, a wasp,etc., such as, a scorpion insectotoxic peptide (Pang (1992)).

(H) An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(I) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, anuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. Examples ofsuch genes include, a callas gene (PCT published applicationWO93/02197), chitinase-encoding sequences (which can be obtained, forexample, from the ATCC under accession numbers 3999637 and 67152),tobacco hookworm chitinase (Kramer et al. (1993)) and parsley ubi4-2polyubiquitin gene (Kawalleck et al. (1993)).

(J) A molecule that stimulates signal transduction. Examples of suchmolecules include, nucleotide sequences for mung bean calmodulin cDNAclones (Botella et al. (1994)), a nucleotide sequence of a maizecalmodulin cDNA clone (Griess et al. (1994)).

(K) A hydrophobic moment peptide. See U.S. Pat. Nos. 5,659,026 and5,607,914, the latter teaches synthetic antimicrobial peptides thatconfer disease resistance.

(L) A membrane permease, a channel former or a channel blocker, such as,a cecropin-β lytic peptide analog (Jaynes et al. (1993)) which renderstransgenic tobacco plants resistant to Pseudomonas solanacearum.

(M) A viral protein or a complex polypeptide derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. See,for example, Beachy et al. (1990).

(N) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Forexample, Taylor et al. (1994) shows enzymatic inactivation in transgenictobacco via production of single-chain antibody fragments.

(O) A virus-specific antibody. See, for example, Tavladoraki et al.(1993), which shows that transgenic plants expressing recombinantantibody genes are protected from virus attack.

(P) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase (Lamb et al. (1992)). The cloningand characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart et al.(1992).

(O) A developmental-arrestive protein produced in nature by a plant,such as the barley ribosome-inactivating gene, have increased resistanceto fungal disease (Longemann et al. (1992)).

Genes That Confer Resistance or Tolerance to a Herbicide

(A) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS (Lee et al. (1988)) and AHAS enzyme (Miki et al. (1990)).

(B) Glyphosate (resistance imparted by mutant EPSP synthase and aroAgenes) and other phosphono compounds such as glufosinate (PAT and bargenes), and pyridinoxy or phenoxy proprionic acids and cyclohexones(ACCase inhibitor encoding genes). See, for example, U.S. Pat. No.4,940,835, which discloses the nucleotide sequence of a form of EPSPsynthase which can confer glyphosate resistance. A Nucleic acid moleculeencoding a mutant aroA gene can be obtained under ATCC accession number39256, and the nucleotide sequence of the mutant gene is disclosed inU.S. Pat. No. 4,769,061. European patent application No. 0 333 033 andU.S. Pat. No. 4,975,374 disclose nucleotide sequences of glutaminesynthase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a phosphinothricinacetyltransferase gene is provided in European application No. 0 242246. De Greef et al. (1989) describes the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al. (1992).

Genes that Confer Resistance or Tolerance to Environmental Stresses

(A) Cold, freezing or frost. This includes genes that code for proteinsthat protect from freezing and for enzymes that synthesizecryoprotective solutes. Examples of such genes are Arabidopsis COR15a(Artus et al. (1996)) and spinach CAP160 (Kaye et al. (1998)). Also inthis category are regulatory genes that control the activity of othercold tolerance genes (Tomashow and Stockinger (1998)).

(B) Drought or water stress. Kasuga et al. (1999) report how stressinducible expression of DREB1A in trangenic plants increases theirtolerance of drought stress. Pilin-Smits et al. (1998) report thatexpression of baterial genes for synthesis of trehalose producestolerance of water stress in transgenic tobacco.

(C) Salinity or salt stress. Genes that code for proteins that minimizeuptake of sodium in the presence of high salt, or cause the plant tosequester sodium in vacuoles, can enable plants to tolerate higherlevels of salt in the soil. The wheat HKT1 potassium transporter,described by Rubio et al. (1999), is an example of the former. Apse etal. (1999) describe how an Arabidopsis Na⁺/H⁺ antiporter can act in thelatter manner.

(D) Metals. Protection from the toxic effects of metals such as aluminumand cadmium can be accomplished by transgenic expression of genes thatprevent uptake of the metal, or that code for chelating agents that bindthe metal ions to prevent them from having a toxic effect. Examples ofsuch genes are Arabidopsis ALR104 and ALR108 (Larsen et al. (1998)) andgenes for the enzymes involved in phytochelatin synthesis (Schafer etal. (1998)).

Genes That Confer or Contribute to a Value-Added Trait

(A) Modified fatty acid metabolism, for example, by transforming maizeor Brassica with an antisense gene or stearoyl-ACP desaturase toincrease stearic acid content of the plant (Knultzon et al. (1992)).

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant, such asthe Aspergillus niger phytase gene (Van Hartingsveldt et al. (1993)).

(2) A gene could be introduced that reduces phytate content. In maize,for example, this could be accomplished by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid (Raboy etal. (1990)).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. Examples of such enzymes include,Streptococcus mucus fructosyltransferase gene (Shiroza et al. (1988)),Bacillus subtilis levansucrase gene (Steinmetz et al. (1985)), Bacilluslicheniformis α-amylase (Pen et al. (1992)), tomato invertase genes(Elliot et al. (1993)), barley amylase gene (Søgaard et al. (1993)), andmaize endosperm starch branching enzyme II (Fisher et al. (1993)).

(D) Modified lignin content. The amount or composition of lignin can bealtered by increasing or decreasing expression of the biosyntheticenzymes for phenylpropanoid lignin precursors, such as cinnamyl alcoholdehydrogenase (CAD), 4-coumarate:CoA ligase (4CL), and O-methyltransferase (OMT). These and other genes involved in formation of ligninare described in Bloksberg et al. (1998).

As those of ordinary skill in the art will recognize, this is only apartial list of possible genes that can be used with the transformationmethod of the present invention. Synthesis of genes suitably employed inthe present invention can be effected by means of mutually priming longoligonucleotides. See, for example, Ausubel et al. (1990) and Wosnick etal. (1987). Moreover, current techniques which employ the polymerasechain reaction permit the synthesis of genes as large as 6 kilobases inlength or longer. See Adang et al. (1993) and Bambot et al. (1993). Inaddition, genes can readily be synthesized by conventional automatedtechniques.

As those of ordinary skill in the art will also recognize, regulatorysequences including promoters, terminators and the like will also berequired, and these are generally known in the art (Zhao et al. (1998)).Plant expression cassettes preferably comprise a structural gene towhich is attached regulatory DNA regions that permit expression of thegene in plant cells. The regulatory regions consist at a minimum of apromoter capable of directing expression of a gene in a plant cell. Thepromoter is positioned upstream or at the 5′ end of the gene to beexpressed. A terminator is also provided as a regulatory region in theplant expression cassette and is capable of providing polyadenylationand transcription terminator functions in plant cells. The terminator isattached downstream or at the 3′ end of the gene to be expressed. Markergenes, included in the vector, are useful for assessing transformationfrequencies in this invention.

The nucleic acid to be delivered to cells may contain selectable markersequences under control of appropriate recognizable promoters for use inselecting transformed cells. Numerous selectable marker genes areavailable for use in plant transformation including, but not limited to,neomycin phosphotransferase II, hygromycin phosphotransferase, EPSPsynthase and dihydropteroate synthase. See, Miki et al. (1993). Othermarkers and promoters are well known in the art. The vectors may alsocontain selectable marker sequences under control of appropriaterecognizable promoters for use in selecting transformed cells. Suitablemarkers and promoters are well known in the art.

The recombinant nucleic acid used for transformation herein may becircular or linear, double-stranded or single-stranded. Generally, thenucleic acid is in the form of a recombinant plasmid and contains codingregions of beneficial heterologous nucleic acid with flanking regulatorysequences which promote the expression of the nucleic acid in the genomeof the transformed plant. For example, the recombinant nucleic acid mayitself comprise or consist of a promoter that is active in othergenotypes, or may utilize a promoter already present in the genotypethat is the transformation target.

The compositions of, and methods for, constructing a nucleic acidsequence which can transform certain plants are well known to thoseskilled in the art, and the same compositions and methods ofconstruction may be utilized to produce the recombinant nucleic aciduseful herein (J. Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press (2d), 1989). The specificcomposition of the nucleotide sequence is not central to the presentinvention and the invention is not dependent upon the composition of thespecific transforming nucleotide sequence which is used.

Restriction enzymes can be introduced, using the method of theinvention, along with linear nucleic acid having compatible cohesiveends to increase the frequency of transformants. The introduction ofrestriction enzymes andn via electroporation has been reported for yeastand Dictyostelium (Schiestl and Petes, 1991; Kuspa and Loomis, 1992).However similar results have not previously been reported with planttransformation.

Selection of Transformed Cells

After beaming, the tissue may be retained on the osmoticum or shortlythereafter transferred to a recovery medium. Following recovery, it isdesirable to identify and select those cells which contain the exogenousnucleic acid. There are two general approaches which have been founduseful for accomplishing this. First, the transformed cells can bescreened for the presence of the recombinant nucleic acid by variousstandard methods which could include assays for the expression ofreporter genes, use of probes for or amplification of the desirednucleotide sequence and assessment of phenotypic effects of therecombinant nucleic acid, if any. Alternatively and preferably, when aselectable marker or reporter gene has been transmitted along with or ispart of the recombinant nucleic acid, those cells which have beentransformed can be identified by detecting expression of the selectablemarker or reporter genes. For example, transient expression may beevaluated by use of the GUS expression cassette containing the GUS gene,which encodes an enzyme for which various chromogenic substrates areknown. Stable transformation may be evaluated by use of the barexpression cassette. Various other selection schemes for identificationof stably transformed tissue may be used, including selection onbialaphos or use of the GFP gene in combination with the Streptomycesbar gene, allowing for visual selection of fluorescing transformedtissues.

Regeneration of Transformed Cells

Conventional regeneration methods, well known in the art, may be usedfor corn species. (Duncan et al., 1985; U.S. Pat. Nos. 5,484,956;5,489,520; 5,177,010; 5,641,664; and 5,350,689, all incorporated hereinby reference.)

Soybean regeneration may be achieved either by the methods of Ranch etal., 1985, or preferably by the methods disclosed herein (Example 9).

EXAMPLES

The present invention is further detailed in the following Examples,which are offered by way of illustration and are not intended to limitthe invention in any manner. Standard techniques well known in the artor the technique specifically described below are utilized.

Example 1 Delivery of Molecules into Cells and Tissues by the Method ofthe Invention

Aerosol droplets containing nucleic acids and/or proteins wereintroduced into target tissue such as cells using the aerosol beamapparatus of the invention (FIG. 1). The aerosol was produced by amicroflow nebulizer such as the HEN from J. E. Meinhard Associates Inc.,or the MCN100 style M4 nebulizer from Cetac Technologies Inc. (Liu andMontaser, 1994; Tan, et al., 1992). The nebulizing gas was high puritycompressed-helium which was regulated with an ACCU-TROL gasregulator—876X model RS-7-4 and filtered through an Arrow F300-02 ITfilter. When HEN and the MCN100 microflow nebulizers were used, thenebulizing pressure was preferably 20-30 psi but worked within the rangefrom about 10 psi to about 40 psi. The entrainment gas filled theentrainment tube and entrained the aerosol droplets in a straight line.Unfiltered, high purity compressed helium was used as the entrainmentgas and was regulated by an Arrow R262 regulator to produce slightpositive pressure as measured by a Gilmont model 65 mm gauge. Theentrainment housing contained a nucleospot to reduce electrostaticcharges and was maintained at a temperature of about

42° C. to about 55° C., and most preferably about 55° C. This reducedcoalescing of the aerosol droplets and was controlled by two OmegaCN9000 series temperature controllers. The sample flow rate to thenebulizer was controlled by a Harvard 11 infusion only syringe pump. Theflow rate was 1 to 1200 μl/min using a sterile Becton Dickinson 1 ccplastic syringe with a 0.2 micron filter attached. The sample contained10 mM Tris buffer (pH 7.0) or a carbohydrate molecule (for example 1 g/lsucrose) and the molecules to be delivered.

As discussed previously, venting of excess aerosol which is reported inU.S. Pat. No. 5,240,842 is not necessary with the method of theinvention. Unexpectedly, venting drastically reduced effectiveness ofthe method of the invention. The target tissue was placed on solidifiedagar medium in a petri dish on the stage, about 3 cm below the nozzletip. The stage was mounted on a XY-4040 precision grade table (NewEngland Affiliated Technologies) which was controlled by LabVIEW 5.0software (National Instruments). The stage moved in a straight line atthe rate of 20-80 mm/sec with about 0.3 mm to 1.0 mm distance betweenpasses. Typically a run of 1.0-3.0 minutes was performed which coveredabout a 2.0 to 4.0 cm diameter area of target tissue. The chamber vacuumwas maintained at around 26 to about 30 inches Hg throughout a given runby a Welch 1405 DuoSeal vacuum pump. The vacuum created a pressuredifferential that was mediated through the nozzle. When the aerosoldroplets produced by the nebulizer in the chamber of comparably highpressure passed through the nozzle, they greatly accelerated into thevacuum of the lower chamber. A small beaker of water was placed in thevacuum chamber to prevent loss of moisture from the target tissue. Thenozzle was preferably a luer-lock 24 gauge (305 um inside diameter)Becton Dickinson syringe needle cut off just proximal to the plasticholder. However, syringe diameters of about 200 to about 500 microns aswell as nozzles known in the art, other than syringes, can be used.

Parameters will vary for particular plant tissues. Tissue which canwithstand the damage caused by the beam may produce more transformantswhen subjected to a more intense barrage of aerosol droplets (producedby using a wider orifice, by slowing stage speed, or by decreasing thedistance between passes, for example).

Briefly, the treatment of target tissue with the aerosol beam apparatuswas performed as follows: 1) place petri dish with tissue on the stageand close vacuum chamber; 2) start the vacuum pump; 3) start the syringepump; 4) set the nebulizing gas pressure; 5) set the entrainment gaspressure, and by this time the correct vacuum in the chamber is reached;and 6) start the movement of the stage and let the system run for thetime needed to complete the run. After the run is completed, shut downthe stage, vacuum, syringe pump, nebulizing gas, entrainment gas, andremove target tissue from the chamber.

Example 2 Introduction of Nucleic Acid into Corn Callus and Detection ofTransient Expression

Embryogenic corn callus of Stine inbred 963 was transferred from stockculture maintenance medium, DN62 (Table 1) to a medium formulated toprovide osmotic stress to the tissue. A preferred embodiment of theinvention, DN62OSM medium was used (Table 1). Preferably the embryogeniccallus was transferred two to three days after transfer to freshmaintenance medium, a time when the cells are dividing rapidly (formaintenance, cultures are routinely transferred every ten days). Afterat least 45 minutes (preferably an hour) and up to 24 hours on thismedium, tissue was collected and oriented in the center of the targetsurface prior to beaming. After beaming, the tissue was allowed toincubate on DN62OSM medium for one day.

The plasmid used in transient expression experiments was preferablyPBARGUS which was obtained from the Plant Gene Expression Center,Albany, Calif., although other plasmids known in the art can be used.Plasmid BARGUS contains a beta-glucuronidase (GUS) expression cassetteconsisting of a GUS gene driven by the corn adh1 promoter and adh1intron1 terminated with a nos terminator, and a bar expression cassetteconsisting of a bar gene driven by a CaMV 35S promoter and an adh1intron terminated with a nos terminator. The GUS expression cassette isused to detect transient expression while the bar expression cassetteconfers tolerance to the herbicide bialaphos. Thus, if desired,selection of stably transformed clones could be achieved. Anotherplasmid used in transient assays was p350096, which has the CaMV 35Spromoter, alcohol dehydrogenase intron six (IV6) driving the GUS genewhich is terminated with the nopaline synthase (nos) 3′ end.Approximately two micrograms of supercoiled plasmid DNA was added to 1.0ml of the buffered solution, however, higher and lower concentrations ofDNA can be used. For example, DNA concentrations as high as about 20μg/ml of DNA and as low as about 0.1 μg/ml to about 1.0 μg/ml wereeffective, although delivery was less effective when using 0.1 μg/ml. Interms of number of DNA molecules, there are approximately 99 billionmolecules in 1 ug of pBARGUS DNA. A DNA concentration of 0.1 ug/mlproduced less transient expression than did 2.0 ug/ml while a DNAconcentration of 1.0 ug/ml produced similar transient expression to 2.0ug. Higher concentrations than 2.0 ug produced marginally more intensetransient expression. Supercoiled or linear DNA could be used intransient expression experiments, however, in this example supercoiledwas preferred because of the ease of preparation. Plasmid DNA wasisolated using Qiagen midi or maxi preps as described by themanufacturer.

The aerosol beam procedure used to transform corn callus was essentiallyas previously described (Example 1). Preferred parameters included anentrainment tube or housing maintained at about 55° C. with the samplesolution flow rate set at about 0.5 ml/hour to about 1.0 ml/hour (8μl/min. to about 17 μl/min.) and the vacuum chamber pressure maintainedat about 29 inches Hg. Treatment of target tissue with the aerosol beamtypically continued for about one to about three minutes, however,beaming can continue for as long as the target tissue can survive beingheld in a vacuum which, in the case of immature corn embryos andembryogenic callus, is at least 3 minutes.

Sterile technique was used routinely to prevent contamination of targettissue. A seventy percent ethanol solution was sprayed on the inside ofthe vacuum chamber and entrainment tube prior to the start of anexperiment and the tubing attached to the nebulizer and the microflownebulizer itself were rinsed out with 70% ethanol which was followed bya wash with sterile water before adding the sample solution. Sterilewater was replaced with the sample solution before treating the targettissue with the aerosol beam apparatus.

Approximately one day after treating the corn callus with the aerosolbeam apparatus, transient expression was evaluated by histochemicalanalysis. Embryogenic callus was incubated in the presence of thesubstrate X-gluc (Gold Biotechnology, Inc.) at a concentration of 0.5mg/ml in 0.1 M sodium phosphate buffer pH 7.0 and 0.1% Triton-x-100 at37° C. After 1-4 hours blue spots appeared indicating GUS expressionwhich verifies that the GUS expression cassette was introduced into thecell with the aerosol beam apparatus. Transient expression results wereused to compare the efficiency of DNA delivery, using equivalent amountsof DNA, to embryogenic callus using either the method of the inventionor a DuPont PDS 1000 particle delivery device. Delivery using the methodof the invention was readily observed to be more efficient in terms ofthe number of color forming units, their size and the intensity of theircolor.

TABLE 1 Medium for Pretreatment, Beaming and Selection Ingredients in 1liter DN62 DN62AG DN62AB DN620SM N6 salts 3.98 g 3.98 g 3.98 g 3.98 g N6vitamins 1 ml 1 ml 1 ml 1 ml Asparagine 800 mg 800 mg 800 mg 800 mgMyo-inositol 100 mg 100 mg 100 mg 100 mg Proline 1400 mg 1400 mg 1400 mg1400 mg Casamino acids 100 mg 100 mg 100 mg 100 mg 2,4_D 1 mg 1 mg 1 mg1 mg Sucrose 20 g 20 g 20 g 20 g Glucose 10 g Sorbitol 45.5 g Mannitol45.5 g AgNO₃ 10 mg 10 mg Bialaphos 1 mg Gelrite 3 g 3 g 3 g 3 g pH 5.85.8 5.8 5.8 DN62B—as for DN62 with the addition of 1 mg/l bialaphosDN62AGB—as for DN62AG with the addition of 1 mg/l bialaphos. N6salts—Sigma Plant Culture Catalogue ref. C1416 N6 vitamins: 2 mg/lglycine, 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine HCl, 1 mg/lthiamine HCl (after Chu C.C. (1978)). The N6 medium and its applicationto anther culture of cereal crops. In: Proc. Symp. on Plant TissueCulture. Sci. Press, Beijing, pp. 43–50.

Example 3 Introduction of Nucleic Acid into Immature Corn Embryos andDetection of Transient Expression

Immature embryos of Stine inbred 963 (10 days' post-pollination) werealso used for transient expression experiments. In this case embryoswere dissected out at between 1 mm and 2 mm in length and either usedimmediately or maintained for up to 10 days, preferably 2 to 3 days, onDN62AG medium (Table 1) prior to beaming. Forty-five minutes beforebeaming the embryos were transferred to DN62OSM. After beaming asdescribed in Example 2 for callus tissue, the embryos were allowed toremain on DN62OSM for 30 minutes before final transfer to DN62AG for oneday.

One day after treating immature embryos with the aerosol beam apparatustransient expression was evaluated by histochemical analysis. Immatureembryos were incubated in the presence of the substrate X-gluc (GoldBiotechnology) at a concentration of 0.5 mg/ml in 0.1 M sodium phosphatebuffer pH 7.0 and 0.1% Triton-x-100 at 37° C. After 1-4 hours blue spotsappeared indicating GUS expression which verifies that the GUSexpression cassette was introduced into the cell with the aerosol beamapparatus. Transient expression results obtained using a DuPont PDS 1000particle delivery device were compared with those obtained using themethod of the invention. Equivalent amounts of DNA were used. Blue spotsappeared more rapidly, were more numerous, larger and more intense usingthe method of the invention.

Example 4 Introduction of Nucleic Acid into Corn Callus and Detection ofStable Transformation

The plasmids used for stable transformation preferably contained the barexpression cassette from PBARGUS, as described previously. This cassetteallowed for selection of a stable transformants using the herbicidebialaphos. Plasmid pRBTBAR contained a Bt expression cassette along withthe bar expression cassette from pBARGUS. Plasmid pBARGFP contained thebar expression cassette along with a gfp expression cassette. In thecase of this plasmid the bar expression cassette was from pSLJ2011 whichwas obtained from The Sainsbury Laboratory, England. The bar gene wasdriven by the CaMV 35S promoter, TMV omega enhancer, and terminated bythe octopine synthase terminator (ocs). The gfp (EGFP) gene was obtainedfrom Clonetech, Inc., and was driven by a CaMV 35S promoter andterminated with a CaMV 35S terminator. For stable transformationexperiments, the DNA concentration varied from 2 ug/ml to 20 ug/ml andthe DNA was either supercoiled or linear. The supercoiled DNA wasisolated as previously described (Example 2). The linear bar expressioncassette was isolated by digesting 20 ug of pBARGUS with HindIII asdescribed by the manufacturer Promega, separated on a 1% agarose gel asis common in the art and extracted from the agarose gel using Qiaex IIas described by the manufacturer (Qiagen). Usually 50% of the DNA foundin a band was recovered which would have been the equivalent in moles to10 ug pBARGUS.

Embryogenic callus, maintained on DN62, is transferred off DN62 1 to 10days, preferably 3 to 6 days, after the previous transfer and placed onDN62OSM for 45 minutes prior to beaming. After beaming as described inExample 1, callus was allowed to remain on this medium for 30 minutes.The beamed tissue was then transferred to DN62B (see Table 1) forselection. Various selection schemes were tested and were successfulincluding selection on bialaphos at concentrations of up to 10 mg/l.Success in selection was enhanced by the use of the gfp gene incombination with the bar gene, allowing for visual selection offluorescing transformed tissues.

Example 5 Regeneration of Stably Transformed Corn Callus

A preferred pathway used to produce stably transformed plants frombeamed embryogenic callus (Stine 963) was performed as follows: afterbeaming on DN62OSM, the callus was transferred to DN62B and cultured fora passage of 14 days; after this period it was transferred again tofurther passages of fresh DN62B medium; after between three and sixpassages on DN62B clones were identified by growth in the presence ofbialaphos (Table 1).

Clones were induced to regenerate plants by selecting actively growingType II callus from clonal tissue, with the objective of obtaining ahigh frequency of so-called “water tower” embryo structures (U.S. Ser.No. 09/203,679 incorporated herein). This type of tissue is preferredbecause regeneration of whole plants is improved. These tissues werethen transferred to DNROB (Table 2). On this medium embryo maturationoccurs. Maturing tissues were then transferred off DNROB after two tothree weeks either to a fresh plate of DNROB or to 0.1 NABA6S (Table 2).After a further one to two weeks, embryos with a shoot meristem wereplaced on MSOG medium (Table 2) or ½MSIBA (Table 2), where germinationoccurs. Plantlets were then transferred to tubes containing ½MSIBAmedium for promotion of further root and shoot development prior tofinal transfer to soil.

TABLE 2 Media for Regeneration Ingredients in 1 liter DNROB 0.1NABA6SMSOG ½MSIBA MS Salts 4.43 g 4.43 g 4.43 g 2.215 g Asparagine 800 mgProline 1400 mg Na2EDTA 37.3 mg 37.3 mg 37.3 mg 37.3 mg Casamino acids100 mg Nicotinic Acid 0.5 mg 1-naphthaleneacetic 0.1 mg acid Abscisicacid 0.1 mg Gibberellic Acid 0.1 mg Indole 3 Butyric 0.1 mg Acid Sucrose60 g 30 g 20 g Sorbitol 20 g Bialaphos 1 mg Gelrite 2 g Phytagar 7 g 7 g7 g pH 5.8 5.8 5.8 5.8

The presence of an expressing bar gene was confirmed in the regenerantsby leaf painting with Liberty. Mendelian ratios of an expressing bargene were routinely observed in the progeny of the regenerants (Table3).

TABLE 3 Target Liberty Liberty Tissue Resistant Sensitive RatioRegenerant* 2-Event B1 Callus 77 23 3:1 Regenerant 8-Event B2 Embryo 6018 3:1 Regenerant 2-Event B3 Embryo 74 25 3:1 Regenerant 1-Event B5Callus 71 26 3:1 Regenerant 7-Event B6 Callus 58 41 1.4:1   *Regenerantnumber refers to the number of the plant regenerated from the indicated“Event”.

Example 6 Introduction of Nucleic Acid into Immature Corn Embryos andDetection of Stable Transformation

Immature embryos of Stine 963 were excised from kernels usually around10 days' post-pollination. At this time the embryos were around 1.0 to1.5 mm in length. Embryos were then placed on DN62AG medium for two tothree days. After this time they were then placed on DN62OSM for 45minutes prior to beaming. After beaming as described in Example 1, theembryos remained on DN62OSM for 30 minutes and then were transferredeither to DN62AG for five days prior to transfer to DN62AGB, or toDN62AGB directly (Table 1).

Example 7 Regeneration of Stably Transformed Corn Embryos

After a further week's culture the corn embryos from Example 6 were thentransferred to DN62AB (Table 1). After several 14-day passages onDN62AB, clones can then be selected. Clonal tissue was induced toregenerate plants according to the description in Example 5.

In one experiment 44 immature embryos were beamed and 5 clones wererecovered (11.3%) which gave rise to transformed plants. The presence ofan expressing bar gene was confirmed in these clones as described inExample 4.

Segregation of expression of the bar gene in Stine elite inbred 963after transformation using the method of the invention is shown in Table3. All regenerants were selfed. Heritability of the nucleic acidinserted by the method of the intention was reflected in the expectedratios for inheritance of a single dominant gene in the progeny oftransformed plants. The fertility of the transgenic plants producedusing the method of the invention is comparable to that ofnontransformed regenerated plants of the same inbred.

Transformation frequency using immature embryos of Stine elite inbred963 and the DuPont PDS-1000 particle gun was around 1% (afterbombardment of several thousand embryos). With the method of the presentinvention, success rates of up to 20% were noted with an average ofaround 3% over all experiments.

Example 8 Improved Growth Rate of Soybean Embryogenic Callus

In another embodiment of the present invention, a novel culture mediamay be used to stimulate high frequency production of embryogenicsoybean callus. Improvement varied with the genotype being cultured. Thelength of time required for a culture passage was unexpectedly reducedto two weeks with the use of this novel medium as compared to four weekstypical with other media. The inclusion of one or more of four mediaconstituents, coconut water, myoinositol, phytic acid and inorganicphosphate concentration, enhanced embryogenic callus production andallowed significant improvements to be made to transgenic cloneproduction in terms of number of clones recovered, embryo morphology,and reduction in the time needed to identify the clones and regenerateplants from them. The medium of Ranch et al., 1985 (referred to hereinas B1-30) was used as the basal medium. An example of the medium of thepresent invention is B1-30 3Co5My0.25PA0.5K (footnote, Table 5).Although this medium is a preferred emobodiment of growth medium, otherconventional media may be utilized in the practice of the invention.

Coconut water has been included in tissue culture media for over 50years. Coconut water is liquid taken from coconuts to promote growth inplant tissue cultures. It is deproteinized then filter-sterilized. Anexample is Sigma Biosciences' Cat. No. C5915. The beneficial effects ofcoconut water were first noted by Overbeek et al., 1941, when it wasfound to permit the growth in culture of heart-stage Datura embryos. Insubsequent years, Steward and others showed that coconut water wascapable of stimulating responses from a variety of plant tissues(Steward et al., 1969). Most commonly coconut water is used atconcentrations of between 5% and 10% by volume of the final culturemedium. The role of coconut water in stimulating the growth ofembryogenic callus in soybean was investigated and it was discoveredthat its effect on cotyledon explants routinely used to initiateembryogenic callus of soybean was detrimental when tested at theseconcentrations. No embryogenic callus was produced in these experiments.However, when coconut water was added to media used for the maintenanceof embryogenic callus, an unexpected beneficial effect was noted interms of rate of growth and quality of somatic embryo morphology. Inview of this result, coconut water was routinely included in media forthe maintenance of embryogenic callus at concentrations of between 3%and 6% by volume of the final culture medium.

One key component of coconut water is myoinositol (Pollard et al.,1961). In an attempt to improve the growth of embryogenic soybean calluswith components of coconut water, it was discovered that myoinositol waseffective in this regard. Myoinositol has been included in tissueculture media for a variety of plant species. Although apparently notrequired for all species (Halperin, 1966), it is routinely included inmedia such as the widely used Murashige and Skoog formulation (Murashigeand Skoog, 1962) at a concentration of 100 mg/l. It is at thisconcentration that it has been included in media used for the culture ofembryogenic callus of soybean (e.g., see Ranch et al., 1985). Unexpectedresults revealed a pronounced and beneficial effect of myoinositol whenused at much higher concentrations (up to 10 g/l) alone or incombination with coconut water.

There are no known reports of the inclusion of phytic acid in culturemedia for any plant species. Soybean seeds are rich in natural chelatingagents, the most prominent of which is phytic acid (Gibson and Ullah,1990). Substantial amounts of phosphate are stored in seeds in the formof phytate (Raboy, 1990). It is possible that the capacity of phyticacid to sequester inorganic phosphate has a significant impact upon Cpartitioning into either starch or sucrose. At 1 gm/l in B1-30,embryogenic callus of a range of genotypes exuded starch, possiblyconfirming the impact of phytic acid on C partitioning. Surprisingly,after this initial effect the soybean callus grew very vigorously andproduced many small globular embryos. Further experiments indicated thatphytic acid at 1, 5, 10, 50, 100, 250, 500 and 1000 mg/l in B1-30significantly increased the rate of growth of embryogenic callus duringthe initial culture passage and/or during maintenance as a stockculture. At 3000 mg/l a clear detrimental effect was observed andembryogenic callus browned and died. Best results over many passageswere obtained with the addition of about 5 mg/l to about 250 mg/l phyticacid to the culture medium depending on the genotype.

Inorganic phosphorous in the form of KH₂PO₄, in excess of the amountconventionally used, was added to the base medium (B1-30) along withmyoinositol and coconut water. This medium was tested against B1-30supplemented with phytic acid in addition to KH₂PO₄, myoinositol andcoconut water. Beneficial effects were noted with 500 and 1000 mg/l bothwith and without phytic acid.

TABLE 4 Growth Media for Soybean* Ingredients in 1 Liter B1-30 B3 B5G MsSalts 4.43 g 4.43 g B5 Salts 3.19 g NaEDTA 37.3 mg 37.3 mg 37.3 mg 2,4-D30 mg Activated 5 g Charcoal Phytagar 8 g 8 g Gelrite 2 g pH 5.8 5.8 5.8*Variations of media referred to in Table 4 were tested, e.g., B1-303Co5My, which was made made by adding 3% coconut water and 5 gm/lmyoinositol to B1-30. Other variations included: B1-30 3Co5My0.25 PA0.5Kwhich contained B1-30 basal medium plus 3% coconut water, 5 gm/lmyoinositol, 0.25 gm/l phytic acid, and 0.5 gm/l additional KH₂PO₄ and ½B5G which contained all ingredients of B5G medium at half strength.

Using the improvements described above, new and effective media weredeveloped for the production of embryogenic soybean callus from avariety of elite genotypes. (See Example 9). The media of the presentinvention are effective for a range of germplasm and also greatly reducethe time required to obtain embryogenic callus in sufficient quantityfor transformation experiments.

Example 9 Culture of Soybean Callus

To initiate cultures, pods were surface sterilized and embryos withimmature cotyledons 3 to 4 mm in length were excised. Individualcotyledons were then cultured on B1-30 medium (Table 4) or on B1-30media containing 100 to 1000 mg/l phytic acid. Typically, a small amountof embryogenic callus proliferated from some of these cotyledonexplants. This callus was then excised and transferred to a B1-30 mediumcontaining between 3% and 6% coconut water or/and between 1 g/l and 10g/l myoinositol. The coconut water and myoinositol requirements foroptimum sustained embryogenic callus growth were found to vary accordingto genotype. Exemplary results using a range of concentrations of thesecomponents are summarized in Table 5.

TABLE 5 Comparative Growth Response Stine Genotype Media Response 96E750B1-30 3Co 10My best 96E750 B1-30 3Co 5My good 96E750 B1-30 3Co good96E750 B1-30 worst 96E692 B1-30 3Co 5My best 96E692 B1-30 6Co 5My v.good 96E692 B1-30 3Co 3My v. good 96E692 B1-30 3Co 1My good 96E692 B1-303Co good 96E692 B1-30 3Co 10My fair 96E692 B1-30 worst 98CB371 B1-30 3Co10My best 98CB371 B1-30 3Co 5My good 98CB371 B1-30 worst 98CB166 B1-303Co 10My best 98CB166 B1-30 3Co 5My good 98CB166 B1-30 worst

In other experiments it was unexpectedly discovered that furtherenhancement of embryogenic callus formation was possible by addition ofabout 1 to about 1000 mg/l of phytic acid and/or additional inorganicphosphate in the form of KH₂PO₄ to B1-30 with myoinositol and coconutwater. The amounts required for improvement vary with genotype. Withthese two additional constituents, suitable amounts of embryogeniccallus for transformation experiments can be obtained within three tosix months, a significant improvement over the year or 18 monthstypically required for elite genotypes on standard media, such as B1-30.

Also effective in preparing tissue for beaming is a brief exposure tococonut water, myoinositol and about 1 gm/l phytic acid for from about 3to 10 days immediately before beaming. Embryogenic callus taken off thismedium and beamed directly grows vigorously after beaming in the periodbefore exposure to a selection agent such as bialaphos (Example 11)allowing for greater survival and growth of transformed cells. Thisimproved survival increases the chances of clone identification duringselection.

Example 10 Introduction of Nucleic Acid into Soybean Callus andDetection of Transient Expression

The apparatus, parameters and beaming method used to introduce nucleicacid into soybean callus were essentially as described in Example 1,unless otherwise indicated in this Example. The sample solutioncontaining DNA was prepared as previously described (Example 2).

Embryogenic soybean callus of Stine 13404-TT was transferred after aculture passage of about 28 to 30 days from stock culture medium (B1-303Co5My 50 mg/l phytic acid—Table 4) to the center of a target platecontaining the same medium. Embryogenic soybean callus can survive beingheld in a vacuum for at least 10 minutes. After one to three days'growth on the target plate, the soybean embryogenic callus is exposed toan aerosol beam of pSLJ4K1 (the 35 S promoter driving the gus gene).After beaming the tissue is spread out on a fresh plate (to minimize therisk of contamination) of the same medium.

Approximately one day after treating the soybean callus with the aerosolbeam apparatus, transient expression was evaluated by histochemicalanalysis. Embryogenic callus was incubated in the presence of thesubstrate X-gluc (Gold Biotechnology, Inc.) at a concentration of 0.5mg/ml in 0.1 M sodium phosphate buffer pH 7.0 and 0.1% Triton-x-100 at37° C. After 1-4 hours blue spots appeared indicating GUS expressionwhich verifies that the GUS expression cassette was introduced into thecell with the aerosol beam apparatus. Transient expression results wereused to compare the efficiency of DNA delivery, using equivalent amountsof DNA, to embryogenic callus using either the method of the inventionor a DuPont PDS 1000 particle delivery device. Delivery using the methodof the invention was readily observed to be more efficient both in termsof the number of color forming units and the intensity of their color.

Example 11 Introduction of Nucleic Acid into Soybean Embryogenic Callusand Detection of Stable Transformation

The plasmids used to stably transform soybean were pSB12BARAHAS andpNPTAHAS. Plasmid SB12BARAHAS contained the bar expression cassette frompSLJ2011 which consisted of a CaMV 35S promoter, TMV omega enhancer, bargene, and nos terminator. Plasmid SLJ2011 was obtained from TheSainsbury Laboratory at The John Innes Center, England. The barexpression cassette was combined with a genomic fragment fromArabidopsis harboring a mutant ahas gene (American Cyanamid). PlasmidpNPTAHAS contained an npt (neomycin phosphotransferase) expressioncassette consisting of a CaMV 35S promoter, TMV omega enhancer, nptgene, and ocs terminator, and the same ahas expression cassettedescribed above. The npt expression cassette was from plasmid SLJ481(Sainsbury Laboratory, John Innes Center, England). The npt expressioncassette provides tolerance to the antibiotic kanamycin and the barexpression cassette provides tolerance to the herbicide bialaphos. Forpurposes of example, supercoiled DNA was used, however, linear DNA ornucleic acid can be expected to work equally well. DNA concentrationvaried from 2 ug/ml to 20 ug/ml. For pSB12BARAHAS approximately 64billion molecules of DNA were delivered per 1 ug.

Embryogenic calluses of several Stine elite varieties, including 96E750,96E94, 97E986, 96E144 and 96E692, were separately collected into thecenter of plates of B1-30 3Co5My or B1-30 3Co5My0.25PA0.5K (Table 4)three days after transfer to fresh medium. The tissue was then beamedwith one of the plasmids described above. After beaming (as described inExample 10) the embryogenic callus was transferred to fresh B1-30 3Co5Myor B1-30 3Co5My0.25PA0.5K for one passage of a month. The tissue wasthen transferred to selective medium containing either 300 mg/lkanamycin or 1 mg/l bialaphos depending on the plasmid used. Withbialaphos, selection typically was maintained at 1 mg/l for the firsttwo one-month passages and then increased to 2 mg/l for the followingthree to seven months. On both bialaphos and kanamycin clones wereidentified after between five and nine transfers on selective medium.Clones were identified when tissue generated by transformationexperiments began to grow vigorously on medium containing a selectiveagent. Once identified, clonal tissue was allowed to increase and wasthen regenerated into plants according to the following protocol: (1)Embryogenic structures were transferred off B1-30 3Co5My or B1-303Co5My0.25PA0.5K to B3 medium (Table 4); (2) after 3 to 4 weeks' growthon this medium clusters of maturing embryos were separated out intoindividual structures and either maintained on the same plate ortransferred to fresh medium; (3) after another 3 to 4 weeks maturingembryos were transferred to B5G medium (Table 4) containing activatedcharcoal and placed in the light; (4) embryos which then elongated andproduced roots were transferred to tubes containing ½ B5G medium (Table4) with no activated charcoal where they continued development intoplantlets; and (5) these plantlets were removed from the tubes andplaced into pots.

Transformation frequency with embryogenic callus of elite Stine soybeanlines was greater using the method of the invention than that achievedin similar experiments using the Dupont particle gun. This result wasconsistent with the results obtained for transient expression inembryogenic soybean callus where grater delivery of DNA was observedusing the method of the invention when compared to the Dupont gun. Thepresence of an expressing bar gene or an expressing ahas gene wasconfirmed by leaf painting (bar) or spraying (ahas). Mendelian ratios ofthe expressing genes were routinely observed in the progeny of theregenerants.

Example 12 Transformation of Bacteria

Transformation of bacteria using the method of the invention wasaccomplished as follows: competent E. coli DH5 alpha (Gibco BRL) orHB101 cells were thawed on ice and 5 to 50 microliters were combinedwith 0.2 ml LB broth. The mixture was pipetted to the center of a plateof LB agar (1.5%) containing 100 mg/l ampicillin and allowed to dry.Aerosol beam parameters were the same as those described previously(Example 1) except as noted in this Example. A pUC-derived plasmidencoding for resistance to the antibiotic ampicillin was delivered tothe bacterial cells, as is known in the art. The concentration of DNAranged from about 1.0 to 1000 μg/ml in 10 mM Tris (pH7.0) or acarbohydrate molecule, such as 1 g/l sucrose. The nebulizing pressurewas from about 10 to about 40 psi, preferably 30 to 40 psi. Theentrainment temperature was maintained in a given experiment and rangedbetween experiments from about 24° to about 55° C., and was preferablymaintained at about 42° to about 55° C. and most preferably at about 55°C. Various sample flow rates were tested ranging from about 0.25 ml/hourto about 2.0 ml/hour (about 4 μl/min. to about 33 μl/min.). Preferablythe flow rate was about 8.0 μl/minute to about 17.0 μl/minute.

The stage on which the bacterial cells were placed was located at adistance of from about 1.5 cm to about 5.0 cm from the end of the nozzlewith a preferred distance of between about 1.5 cm and about 3.2 cm. Thespeed at which the stage moved during beaming was adjusted to about 10mm/sec to about 100 mm/sec with a preferred speed of about 20 mm/sec toabout 100 mm/sec. The distance between passes was varied from about 0.2mm to about 1.0 mm. Most preferable distance between passes was about0.3 mm to about 0.4 mm.

The vacuum in the sample chamber was varied from about 26 psi to about30 psi with best results obtained at the higher vacuum pressures, forexample 29 psi. Nozzles with orifices of about 254 to 500 microns wereused. Preferable were nozzles with diameters of about 300 to about 330microns.

After beaming, plates were incubated at 37° C. for 15 to 20 hours. Nogrowth was observed in the control without DNA, however, when DNA wasintroduced, growth of thousands of transformed colonies was evident. Asa control, the same sample of DNA which was beamed was sprayed on thecells using the same microflow nebulizer used with the aerosol beamapparatus but with no supersonic acceleration of aerosol droplets. Notransformed colonies resulted from this treatment. As an additionalcontrol, bacteria were bombarded with tungsten particles coated with thesame pUC-derived plasmid using the particle gun and a protocolessentially as described by Smith et al. (1992). This protocol producedno transformants. The protocol of Smith et al. (1992), was altered inthe practice of the present invention in that the relative humidity wasnot adjusted nor was osmoticum used.

Example 13 Use of the Method of the Invention to Increase TransformationFrequency

Transformation frequency in plants can be increased by introducingrestriction enzymes simultaneously with the transforming DNA using themethod of the present invention. Parameters for the aerosol beamapparatus are as described previously for plant cells (Example 1). Cornand soybean target tissue are prepared as described previously (Examples4 and 5). For selection of transformants, a number of differentselective agents can be used including, but not limited to, bialaphos,kanamycin, hygromycin, and imazapyr. By way of illustration, selectionusing imazapyr and mutant AHAS genes is described.

Plasmid pCD220 carries a mutant corn AHAS gene. Expression of this genein corn confers resistance to the herbicide Arsenal (active ingredientimazapyr) manufactured by American Cyanimid. The plasmid is digestedwith the restriction enzyme Xba I as described by Promega. Thelinearized DNA fragment carrying the gene and regulatory elementsnecessary for expression in corn are separated from the remainingplasmid DNA in a 1% agarose gel as practiced in the art. The genomic DNAis then purified from the gel using the QIAEX II gel extraction kitaccording to the manufacture (Qiagen). About 10 micrograms of linear DNAwith Xba I compatible ends and 200 units of Xba enzyme are combined in 1ml of 10 mM Tris pH 7.0, or in 1 ml of a buffer solution recommended forXba I by the manufacturer (Promega). The solution containing the DNA andthe enzyme is then beamed into corn cells as previously described.Transformants are selected as described previously. The simultaneousintroduction of DNA with the appropriate restriction enzyme cansignificantly increase the frequency of transformation.

A mutant AHAS gene from Arabidopsis can be introduced into soybean cellsusing the same methods. Plasmid AC321 is digested with Xba I asdescribed above for plasmid pCD220. This Arabidopsis genomic fragment isthen used for transformation of soybean together with the Xba Irestriction enzyme. Again, the number of transformants obtained usingthe enzyme together with the DNA can be significantly higher than whenusing the DNA alone.

Example 14 Introduction of Carbohydrate and Plant Growth Regulator intoCells

Glucose and 2,4-D were introduced into cells of cultured immatureembryos of Stine corn inbred 963 by the method of the present invention.Preparation of embryos and parameters for the aerosol beam apparatuswere the same as described for Example 6. Glucose was used at aconcentration of 1 gm/l. 2,4-D was used at a concentration of 0.1 mg/l.After beaming with pBARGUS, glucose, and 2,4-D, stably transformedclonal tissue was selected and plants regenerated as described inExample 7. A significant increase (2 to 10 fold) in the number of clonesrecovered was noted from embryos beamed with glucose, 2,4-D and DNA whencompared with embryos beamed with DNA alone. Similar results wereobtained using 1 g/l sucrose in place of 1 g/l glucose.

Example 15 Introduction of Protein into Cells

The protein glucuronidase was introduced into embryogenic callus cellsof Stine corn inbred 963 by the method of the present invention. Targetpreparation and aerosol beam parameters were as described in Example 2.Glucuronidase from Sigma (cat # G2035) was used at a concentration of320 μg/ml in 10 mM Tris (pH 7.0). After treating of corn callus with theaerosol beam apparatus, the tissue was immediately incubated in X-glucsubstrate, as described in Example 2. After 1 to 4 hours blue spotsappeared indicating the intracellular presence of GUS. The intracellularpresence of GUS was then confirmed by microscopic examination. As acontrol, protein was applied to the surface of the embryogenic calluswith an atomizer and the callus was then incubated for about 1 to 4hours in x-gluc substrate. No blue spots were observed with thistreatment.

Example 16 Introduction of Nucleic Acid Together with Other Moleculesinto Cells

Nucleic acid and protein were simultaneously delivered into bacterialcells. Preparation of bacteria and parameters for the aerosol beamapparatus were the same as described previously for delivery of DNA intobacteria (Example 12). The same buffer, plasmid, and plasmidconcentration was used as described in the transformation of bacteria.Ribonuclease A (RNASE A) (Sigma cat. no. R6513) was added to the DNAsample at a final concentration of 2.5 μg/ml. After the sample of DNAand RNASE A was beamed into bacteria, no colonies were observed onmedium containing ampicillin indicating the cytotoxic effect of RNASE Ain the cells.

As a control, RNASE A at the same concentration used for beaming waspipetted on the cells after the cells were beamed with DNA only. Here,transformed colonies grew indicating that the RNASE A was not taken upnaturally by the cell. To verify that the RNASE A was not degrading theplasmid, the plasmid treated with RNASE A was run on a standard 1%agarose gel. After staining the DNA with ethidium bromide, the DNAappeared intact and no degradation was evident. As a further control,RNASE A was inactivated with diethylpyrocarbonate (DEP). Inactivationwas carried out as follows: 2.5 micrograms of RNASE A was added to 1.0ml 10 mM Tris (pH 7.0), then 4.0 μl of DEP was added. This mixture wasvortexed and incubated for 2-4 hours at room temperature, after whichtime, gases were evacuated from the tube by heating the mixture to 85°C. for 15 minutes with the cap of the tube off. DNA was then added andthe sample beamed into cells. The introduction of DNA and inactivatedRNASE A resulted in the normal transformation of bacteria. Thus, theaddition of RNASE A to the DNA did not prevent the DNA from entering thecell. From these experiments it follows that both DNA and protein weredelivered together into bacterial cells.

Example 17 Optimization of Sample Flow Rate for Bacteria

Transformation of bacteria using the method of the invention wasperformed as described in Example 12. The sample flow rate was variedfrom about 1 μl/minute to about 200 μl/minute. Although sucrose was usedin this example, other carbohydrates such as glucose can be used in thepractice of the invention. The efficiency of DNA delivery is reflectedin Table 6 as number of bacterial colonies able to grow on agarcontaining 100 mg/l ampicillin at the listed sample flow rates.

The preferred sample flow rate for introduction of DNA into bacteria wasabout 1 μl/minute to about 200 l/minute, a more preferred flow rate wasabout 4 μl/minute to about 50 μl/minute and the most preferred flow ratewas about 8 μl/minute to about 17 μl/minute.

Example 18 Optimization of Sample Flow Rate for Corn

Introduction of DNA into immature corn embryos was performed using themethod of the invention as described in Example 3. The sample flow ratewas varied from about 1 μl/minute to about 200 μl/minute. Transientexpression results were used to compare the efficiency of DNA delivery.Results are reflected in Table 6 as number of blue spots per embryo atthe listed sample flow rates. The preferred sample flow rate forintroduction of DNA into corn is about 1 μl/minute to 200 μl/minute, amore preferred rate is about 4 μl/minute to about 50 μl/minute and amost preferred rate is about 8 μl/minute to about 50 μl/minute.

Example 19 Optimization of Sample Flow Rate for Soybean

Introduction of DNA into soybean callus was performed using the methodof the invention as described in Example 10. The sample flow ratesvaried from about 1 μl/minute to about 200 μl/minute. Transientexpression results were used to compare the efficiency of DNA delivery.Results are reflected in Table 6 as number of blue spots per plate atthe listed sample flow rates. The preferred sample flow rate forintroduction of nucleic acid into soybean callus was about 1 μl/minuteto about 200 μl/minute, with a more preferred rate of about 4 μl/minuteto about 50 μl/minute and a most preferred rate of about 8 μl/minute toabout 50 μl/minute.

TABLE 6 Flow Rate in Microliters Per Minute* 1.0 2.0 4.0 8.0 17.0 50.0100.0 200.0 Bacteria 0 0 24 236 179 91 2 2 Corn <1.0 1.0 4.0 7.5 9.0 8.01.0 <1.0 Soybean 0 0 139 264 249 211 65 22 *The effect of sample flowrate on the introduction of DNA into bacteria, corn, and soybean wasdetermined as described in Examples 17, 18 and 19 for the transformationof bacteria, corn, and soybean, respectively. The results in Table 6represent a typical experiment. For bacteria, the efficiency of DNAdelivery was determined by the number of transformed bacterial coloniesper plate growing on antibiotic containing medium. For corn theefficiency of DNA delivery was determinedby the number of blue spots perembryo, and for soybean the number of blue spots are per plate.

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than a limiting sense, as it is contemplated that modificationswill readily occur to those skilled in the art, within the spirit of theinvention and the scope of appended claims.

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1. A method for the introduction of one or more carbohydrates into a plant cell or a bacterial cell, wherein the method comprises: (a) preparing a solution containing said carbohydrates; (b) supplying said solution for conversion to aerosol droplets which are produced by a microflow nebulizer at a flow rate of between about 1 μl/minute to about 100 μl/minute; (c) producing aerosol droplets comprising said carbohydrates; (d) accelerating by jet expansion of an inert gas said aerosol droplets toward said cell wherein said cell is in a vacuum; (e) impacting said cell with said accelerated aerosol droplets; and (f) producing a cell containing said carbohydrates.
 2. The method of claim 1, wherein said cell is a bacterial cell.
 3. The method of claim 1, wherein said plant cell is a monocotyledonous plant cell.
 4. The method of claim 3, wherein said monocotyledonous plant cell is a corn cell.
 5. The method of claim 1, wherein said plant cell is a dicotyledonous plant cell.
 6. The method of claim 5, wherein said dicotyledonous cell is a soybean cell.
 7. The method of claim 1, wherein said aerosol droplets are continuously targeted toward said cell.
 8. The method of claim 1, further comprising the placement of said cell on a target surface the linear and rotational movement of which can be separately controlled.
 9. The method of claim 1, wherein said method further comprises regenerating a plant from said impacted plant cell.
 10. The method of claim 4, wherein the said flow rate is between about 4 μl/minute and about 50 μl/minute.
 11. The method of claim 10, wherein the said flow rate is between about 8 μl/minute and about 50 μl/minute.
 12. The method of claim 6, wherein the said flow rate is between about 4 μl/minute and about 50 μl/minute.
 13. The method of claim 12, wherein the said flow rate is between about 8 μl/minute and about 50 μl/minute.
 14. The method of claim 1, wherein the said flow rate is between about 4 μl/minute and about 50 μl/minute.
 15. The method of claim 14, wherein the said flow rate is between about 8 μl/minute and about 50 μl/minute.
 16. The method of claim 1, wherein said one or more carbohydrates is sucrose. 